This invention lies in the field of liquid-liquid partition chromatography, and in particular in the chiral separation of enantiomers using chromatographic techniques.
Countercurrent chromatography (CCC) is a form of liquid-liquid partition chromatography which relies on the continuous contact between two immiscible solvents, one of which is mobile relative to the other, in a flow-through tubular column, free of any solid support matrix. The retention time of a solute in the phase contact region of the system is determined by the volume ratio of the solvents, the partition coefficient of the solute between the solvents, and the degree of contact between the solvents. Like other forms of liquid-liquid partition chromatography, one of the solvents serves as a carrier, drawing the solutes from the other solvent and carrying the solutes out of the column in the order of elution. This carrier solvent is thus referred to as the mobile phase, while the other solvent is referred to as the stationary phase, even though it is not strictly stationary in many applications of the method. Solvent mixing, retention of the stationary phase in the column, and solute partitioning all take place in the column by the aid of a suitable acceleration field established by gravity, centrifugal force or both, and the configuration of the column.
Most equipment used for CCC separations involves a coil of column tubing, a portion of which is filled with the stationary phase while the mobile phase is passed through it. By varying the length and diameter of the tubing, CCC has been used for both analytical and preparative separations.
The flow rate of the mobile phase may be varied by varying the field imposed on the column. Units which operate in the presence of a gravitational field only are restricted to slow flow rates, with the resulting separations typically requiring 1 to 3 days, to avoid displacing the stationary phase. A unit which operates in the presence of a centrifugal acceleration field of 40 g or more allows faster flow rates and permits separation times of only a few hours.
Separations by CCC may be performed using any immiscible pair of solvents, provided that the solvents differ in density to at least a slight degree. Both normal-phase and reverse-phase separations may be performed, with the more polar solvent as the stationary phase for normal-phase separations, and the less polar solvent as the stationary phase for reverse-phase separations.
The operational aspects of CCC are similar to the more conventional liquid-liquid chromatography (LLC). Typically, after the immiscible solvent phases are equilibrated relative to one another, the column is filled with the stationary phase. The sample is then injected into the column and elution with the mobile phase is begun. The centrifuge is then started and the eluting fractions are collected. Initially, the fractions are composed of the stationary phase which is displaced from the column. However, once hydrodynamic equilibrium between the phases is achieved, only small portions of the stationary phase will co-elute with the mobile phase. The effluent is continuously monitored with a uv detector and fractionated into test tubes using a fraction collector. The collected fractions are monitored by any of a variety of means including spectroscopic methods and thin-layer chromatography.
Countercurrent chromatographic theory, as well as apparatus for performing the method, are described by Ito, Y., in xe2x80x9cPrinciple and Instrumentation of Countercurrent Chromatography,xe2x80x9d in Countercurrent Chromatography: Theory and Practice Mandava, N. B., and Ito, Y., eds., pp. 79-442 (Marcel Dekker, New York, 1988) and by Conway, W. D., in Countercurrent Chromatography: Apparatus, Theory and Applications (VCH, New York, 1990). Most countercurrent chromatographs use a column which is formed into a helical coil. This coil is in turn mounted onto a column holder in various configurations relative to the means for rotating it and relative to the acceleration field that acts on it.
Each column and each type of rotation produce different types of mixing between the solvent phases and are particularly suited for specific separations. However, certain disadvantages to CCC exist.
One disadvantage associated with CCC is the increased peak width associated with increased retention time of the solute. This increased peak width makes detection of the solute more difficult, and requires a larger volume of eluate to be collected and processed in order to obtain a maximum yield of solute. This disadvantage is particularly acute when preparative separations are desired. Nevertheless, increased retention time is desirable in order to avoid coeluting impurities with the solute. Commonly-owned, copending U.S. patent application Ser. No. 07/946,613, filed Sep. 18, 1992, discloses a method for obtaining sharp elution peaks in analytical or semi-preparative CCC without decreasing the retention time of the solute, by adding a peak sharpening agent to either the stationary phase or the sample mixture. When acidic compounds are to be separated, the peak sharpening agent is an acid. When basic solutes are to be separated, the peak sharpening agent is a base.
More recently, an unusually efficient separation of mixtures of acids or bases has been described using a unique modification of the techniques of countercurrent chromatography. See, Ito, et al. U.S. Pat. No. 5,332,504, the disclosure of which is incorporated herein by reference. According to this modification, the two immiscible liquid solutions which are to serve as the stationary and mobile phases, respectively, are modified prior to the performance of the separation by rendering one of the phases acidic and the other basic. Separation of a mixture of acids is then performed in a system in which the acidified solution serves as the stationary phase and the basified solution as the mobile phase. Conversely, separation of a mixture of bases is performed in a system in which the basified solution serves as the stationary phase and the acidified solution as the mobile phase. Individual acid or basic solutes separated by this method elute in contiguous, well-resolved, rectangularly shaped peaks, the solutes eluting in order of their PKa values and hydrophobicity and the fractions within any single peak being of substantially constant concentration. The combined fractions within each peak differ in pH, successively increasing in the case of a basic mobile phase and successively decreasing in the case of an acidic mobile phase. For this reason, the technique has been referred to as xe2x80x9cpH-zone-refining countercurrent chromatography. xe2x80x9d
A recent modification of pH-zone-refining countercurrent chromatography is carried out in a manner analogous to displacement chromatography. See, commonly-owned, copending U.S. patent application Ser. No. 08/263,924, filed Jun. 21, 1994 and incorporated herein by reference. This method uses a retainer base (acid) in the stationary phase to retain analytes in the column and a displacer acid (base) to elute the analytes in the decreasing (or increasing) order of pKa and hydrophobicity. The elution produces a train of highly concentrated rectangular solute peaks with minimum overlap. To use pH-zone-refining CCC in a displacement mode, the mobile and stationary phases are switched. Thus, the original eluent becomes a retainer to retain analytes in the stationary phase, and the original retainer acid becomes a displacer to displace the analytes from the stationary phase to the mobile phase at the back of the solute bands.
Displacement countercurrent chromatography and pH-zone-refining countercurrent chromatography (in the normal mode) both entail certain advantages over previously known counter-current chromatography techniques. First, the method permits one to load the sample as a suspension into the separation column. Thus, mixtures of compounds that are only partially soluble in the solvent system can be separated efficiently. In addition, the lack or small degree of elution peak overlap permits one to separate mixtures of greater volume than before in any given column without loss of resolution. For example, columns which are otherwise recommended for separations of mixtures of a certain maximum size can be used for separating mixtures up to ten times that size or greater. Likewise, mixtures containing higher concentrations of the acid or basic solutes can be separated with no loss in resolution. As the concentration of solute increases, the separation simply produces a wider plateau for each solute.
With an increasing demand for optically active compounds, the development of methods for the separation of enantiomers is being intensively pursued. The preparation of optically active compounds has become very important for the development of new biologically active substances containing one or several chiral centers, because many chiral drugs display different activity and toxicity profiles with respect to their absolute configuration.
The direct separation of enantiomers by chromatography is now widely used and a large number of chiral columns using a solid support chiral stationary phase becomes more and more popular and diversified. More than one hundred chiral stationary phases are commercially available allowing many analytical problems to be solved. However, few preparative applications have been reported because of the limited capacity of the standard size columns. Large columns are very expensive.
Compared to the rapid development of optical isomer separation by liquid column chromatography using chiral stationary phases, little work has been reported concerning the separation of optical isomers by countercurrent chromatography. Recently, others have successfully separated D,L-amino acid derivatives by centrifugal partition chromatography. See, Oliveros, et al., J. Liq. Chromatogr. 17:2301 (1994). However, this method can only be applied to microgram quantities of samples. Moreover, the chromatographic fractions isolated by Oliveros, et al. were contaminated with substantial amounts of chiral selector and further purification was required. This may also be a problem in other liquid-liquid chromatography techniques.
The present invention provides methods for the preparative-scale separation of optical isomers of a racemic pair using high-speed countercurrent chromatography (HSCCC). In one embodiment, a chiral selector is held in a liquid stationary phase through which a mobile phase flows, the chromatographic process taking place between the two liquid phases. The separations are carried out with a two-phase solvent system in which the chiral selector is distributed almost exclusively in the stationary phase while the analytes are partitioned between the two phases. The column is first filled with the stationary phase containing the chiral selector, followed by sample injection. The mobile phase is then eluted through the column. A racemic mixture of enantiomers is resolved according to the difference in affinities of the D- and L-forms (or (+) and (xe2x88x92) forms) to the chiral selector. As is a common practice in high performance liquid chromatography (HPLC), the CCC separation can be repeated by successive sample injection without renewing the column contents. The advantage of the present method is derived from the fact that the column contains no solid support and there is no need to immobilize the chiral selector to the solid stationary phase which involves a complicated synthetic process. The liquid stationary phase can hold a large amount of the chiral selector compared to the solid support chiral stationary phase within the conventional chromatographic column. The sample loading capacity and resolution of racemates depend not only on the column volume but also on the concentration of the chiral selector in the stationary phase. Consequently, in HSCCC the chiral separation can be applied in both analytical and preparative scales using the same column only by adjusting the concentration of chiral selector in the stationary phase.
The present invention also provides methods for the preparative-scale separation of optical isomers of a racemic pair using pH-zone-refining countercurrent chromatography.